Guramrit Singh

A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay.

The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1–3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3′ UTR–associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3′ UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3′ UTR. Interestingly, while an artificial 3′ UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3′ UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon.

Upf1 ATPase-Dependent mRNP Disassembly Is Required for Completion of Nonsense- Mediated mRNA Decay

Cellular mRNAs exist in messenger ribonucleoprotein (mRNP) complexes, which undergo transitions during the lifetime of the mRNAs and direct posttranscriptional gene regulation. A final posttranscriptional step in gene expression is the turnover of the mRNP, which involves degradation of the mRNA and recycling of associated proteins. How tightly associated protein components are released from degrading mRNPs is unknown. Here, we demonstrate that the ATPase activity of the RNA helicase Upf1 allows disassembly of mRNPs undergoing nonsense-mediated mRNA decay (NMD). In the absence of Upf1 ATPase activity, partially degraded NMD mRNA intermediates accumulate in complex with NMD factors and concentrate in processing bodies. Thus, disassembly and completion of turnover of mRNPs undergoing NMD requires ATP hydrolysis by Upf1. This uncovers a previously unappreciated and potentially regulated step in mRNA decay and raises the question of how other mRNA decay pathways release protein components of substrate mRNPs.

New insights into the formation of active nonsense-mediated decay complexes

In the nonsense-mediated mRNA decay (NMD) pathway, an exon-junction protein complex (EJC) and hUpf proteins mediate rapid downregulation of aberrant mRNAs that terminate translation upstream of the last splice junction. Two EJC subunits, Y14 and RNPS1, have been proposed to act as a link between splicing and NMD by recruiting hUpf3 and the other hUpf proteins. New studies now present evidence that Y14 is directly involved in NMD, and that Y14 is required for hUpf3 activity. These findings suggest unforeseen intricacies in the formation of active NMD complexes.

RIPiT-Seq: a high-throughput approach for footprinting RNA:protein complexes.

Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds.

Isolation and characterization of a novel Bacillus strain from coffee phyllosphere showing antifungal activity

Aims: The isolation and characterization of a novel coffee‐associated Bacillus mojavensis strain, designated as strain AB1, and its survival on the coffee phyllosphere.

ASPeak: an abundance sensitive peak detection algorithm for RIP-Seq

SUMMARY Unlike DNA, RNA abundances can vary over several orders of magnitude. Thus, identification of RNA-protein binding sites from high-throughput sequencing data presents unique challenges. Although peak identification in ChIP-Seq data has been extensively explored, there are few bioinformatics tools tailored for peak calling on analogous datasets for RNA-binding proteins. Here we describe ASPeak (abundance sensitive peak detection algorithm), an implementation of an algorithm that we previously applied to detect peaks in exon junction complex RNA immunoprecipitation in tandem experiments. Our peak detection algorithm yields stringent and robust target sets enabling sensitive motif finding and downstream functional analyses. AVAILABILITY ASPeak is implemented in Perl as a complete pipeline that takes bedGraph files as input. ASPeak implementation is freely available at https://sourceforge.net/projects/as-peak under the GNU General Public License. ASPeak can be run on a personal computer, yet is designed to be easily parallelizable. ASPeak can also run on high performance computing clusters providing efficient speedup. The documentation and user manual can be obtained from http://master.dl.sourceforge.net/project/as-peak/manual.pdf.

The exon junction complex: a lifelong guardian of mRNA fate.

During messenger RNA (mRNA) biogenesis and processing in the nucleus, many proteins are imprinted on mRNAs assembling them into messenger ribonucleoproteins (mRNPs). Some of these proteins remain stably bound within mRNPs and have a long‐lasting impact on their fate. One of the best‐studied examples is the exon junction complex (EJC), a multiprotein complex deposited primarily 24 nucleotides upstream of exon–exon junctions as a consequence of pre‐mRNA splicing. The EJC maintains a stable, sequence‐independent, hold on the mRNA until its removal during translation in the cytoplasm. Acting as a molecular shepherd, the EJC travels with mRNA across the cellular landscape coupling pre‐mRNA splicing to downstream, posttranscriptional processes such as mRNA export, mRNA localization, translation, and nonsense‐mediated mRNA decay (NMD). In this review, we discuss our current understanding of the EJC’s functions during these processes, and expound its newly discovered functions (e.g., pre‐mRNA splicing). Another focal point is the recently unveiled in vivo EJC interactome, which has shed new light on the EJCs location on the spliced RNAs and its intimate relationship with other mRNP components. We summarize new strides being made in connecting the EJC’s molecular function with phenotypes, informed by studies of human disorders and model organisms. The progress toward understanding EJC functions has revealed, in its wake, even more questions, which are discussed throughout. WIREs RNA 2017, 8:e1411. doi: 10.1002/wrna.1411

The Human Nucleolar Protein FTSJ3 Associates with NIP7 and Functions in Pre-rRNA Processing

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A′ to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.

The exon junction complex undergoes a compositional switch that alters mRNP structure and nonsense-mediated mRNA decay activity

The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that EJC originates as an SR-rich mega-dalton sized RNP that contains RNPS1 but lacks CASC3. After mRNP export to the cytoplasm and before translation, the EJC undergoes a remarkable compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general whereas CASC3 is needed for NMD of only select mRNAs. The promotion of switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.